Bone and teeth are known to contain a factor (or factors) which has the capacity to direct commitment of primordial mesenchymal cells towards cartilage and bone formation. Implantation of appropriately decalcified bone or dentin matrix into a soft tissue such as a muscle pouch induces bone formation through a process akin to endochondral ossification. (1) Perivascular mesenchymal cells migrate to the implant and differentiate into cartilage, which then is replaced by true bone. (2)
Koskinen, et al (3) have developed a cell culture assay to detect the presence of chondrogenic activity. This assay is capable of indicating the presence of a chondrogenic factor at the nanogram level. The assay utilizes fibroblast-like cells grown from explants of neonatal rat muscle. These cells can be induced to change to a chondrocyte-like mode of expression. The transformation is followed by assays for cartilage-specific glycosaminoglycan (proteoglycan) and Type II collagen production.
Urist et al. (4) have proposed that the inducing factor in bone is a protein, which they called bone morphogenetic protein (BMP). BMP is reported to be anionic with a relative molecular weight of about 18,500. (4, 8) It can be extracted from an acid demineralized bone matrix with 4.0M guanidine HCl or 6.0M urea+0.5M CaCl.sub.2, but the yields are low. Chondrogenic/osteogenic inductive agents have been very difficult to isolate and purify. The absolute amount of these biofactors is very small. Further, they are highly interactive with other bone proteins during isolation, and the assays for their activity have required substantial quantities of the factor.
In 1985, Seyedin et al. reported the characterization of two cartilage-inducing factors derived from bovine demineralized bone. (5) These factors were called CIF-A and CIF-B. Their isolation was by a sequence of extraction, gel filtration, cation-exchange chromatography, and reverse phase HPLC. Both factors were described as having an apparent molecular weight (M.sub.r) of around 26,000. It was further reported that in the presence of 2-mercaptoethanol CIF-A and CIF-B converted to species of about one-half M.sub.r, indicating that CIF-A and CIF-B were probably dimers. Subsequently, Seyedin et al. (6, 7) reported that CIF-A and CIF-B were similar if not identical to the transforming growth factor, (TGF-.beta.) as based on partial sequencing. As pointed out by Seyedin et al. (5), others have reported the extraction of cartilage induction proteins from bone.
Sommerman et al. (9) have reported that human dentin matrix induces cartilage formation in vitro by mesenchymal cells derived from embryonic muscle. Heretofore, however, no one has reported the extraction and purification of the chondrogenic factor in teeth. Other components of dentin matrix have been isolated. Kuboki, et al. (10) reported the preparation of a dentin phosphoprotein, and Butler et al. (11) described the isolation and partial characterization of dentin proteins and proteoglycans. Butler et al. (11) used a preparative method including CaCl.sub.2 -precipitation of the EDTA extract of dentin, followed by dialysis again of the resultant supernatant, and then passage of the supernatant through a Sephadex G-50 column. A high molecular weight fraction was obtained.
Tsay and Veis (12) have described the preparation of phosphophoryn components of rat incisor dentin, using successive steps of CaCl.sub.2 precipitation, ion-exchange chromatography, and gel filtration. The phosphophoryn obtained had a relative molecular weight (M.sub.r) of around 90,000. That procedure utilized the precipitate from a CaCl.sub.2 precipitation of the dentin extract rather than working with the supernatant. No chondrogenic factor was prepared.